This proposed Phase I SBIR project will transfer RASL-Seq, a targeted sequencing technology developed in academia, to a small business, 255Xpress, Inc., and demonstrate feasibility for its commercialization as a platform for drug discovery and high throughput screening (HTS). The technology detects hundreds of RNA targets at once in cell lysates, using oligo ligation, amplification, and next-generation sequencing for quantitation. This highly multiplexed HTS assay measures sets of genes and pathways, rather than single targets, and expresses the result as a single score. As a result, drug responses can be detected without monitoring the druggable target, and the technology can be used to assess efficacy and side effects at the same time. This multiplexed profiling approach will enable the identification of off-target adverse effects early in the drug discovery process, rather than being discovered late in clinical development or post marketing where the only option is to stop development or withdraw the drug. Given that 1/3rd of late stage failures are due to unanticipated off- target effects that could have been avoided, RASL-Seq will have a significant impact across all therapeutic areas, increasing the efficiency, cost, and success of getting drugs to market. To demonstrate commercial feasibility, we intend to develop robust probe design and data analysis methods, hardened reagent formulations and assay processes, and internal and external assay controls. We will establish performance measures (reproducibility, dynamic range, mis-ligation rates, and the behavior of controls), and conduct robustness and verification studies. We will also demonstrate the feasibility of HTS and QSAR optimization assays measuring the expression of 10 to 1,000 genes with reproducibility CV's of <15%. RASL-Seq can be fully automated and permits the pooling of samples before sequencing, which we will demonstrate will reduce total cost per sample for HTS to less than $3 per sample, a price point that is critical for commercial adoption of multiplexed assays. RASL-Seq can also be used to repurpose drugs, or to salvage programs where the leads fail in clinical development due to off-target effects, by profiling to identify a signature and then re-testing analogs and re-screening. There is an acute need for new targets for drug mechanisms of action, and this technology platform provides the capability to unlock previously undruggable mechanisms of action to rational drug discovery.